text: A Type IF-3 CAST (Tn6677) was initially identified in Vibrio Cholerae and has been extensively studied. This system contains proteins TnsA, TnsB, and TnsC that complex with Cas6, Cas7, and a Cas5-Cas8 fusion through interactions with TniQ. Initial integration steps include TniQ-Cascade binding at the target site and TnsA and TnsB excision of the transposon, which is followed by TnsC binding to TniQ and transposase binding to TnsC. There can be off-targeting prior to this final step, but TnsB and TnsC binding leads to a final proofreading step to maintain a high on-target percentage. Tn6677 integration has been validated at near 100% on-target efficiency at site specific locations in multiple points in the host genome. Other systems have also been characterized and validated in this class with varying ranges of efficiency, and include orthogonal systems for multiplexed insertions up to 10kb.
  A unique characteristic of Type IF-3 systems is the presence of self-targeting guide RNA that are used to target the host chromosome. These systems have privatized the corresponding spacers through the use of atypical crRNA that prevent endogenous Type 1F systems from using the guides and their interference mechanisms to degrade the host. Another privatization mechanism is the use of mismatch tolerance allowing only CAST systems to target locations in the genome without an exact match to the spacer.